Legumes developed symbiotic associations with rhizobia to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. One microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced to unravel the miRNA mediated regulation of symbiosis in chickpea. Sequencing of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. Phylogenetic analysis of the precursor sequences of the 91 miRNAs clearly indicated a clustering of two distinct miRNAs in the same clade representing a close ancestral precursor. Further, biogenesis of miRNAs was predicted using the miRNAs identified from different legume genomes. The miRNA reads from the nodule library were also mapped onto bacterial genomes from which bacterial small RNA were predicted. The antagonistic expression of some of the miRNA-target pairs was investigated and the negative co-related expression profiling proved the validity of the libraries and the miRNA-target pairs. Four miRNAs were selected based on the antagonistic expression profiling and were ectopically expressed in chickpea roots by hairy root transformation. The overexpression lines showed significant change in nodule numbers. The target of miR171f (NRK), miR394 (HP) and miR1509 (AK) are novel ones being reported for the first time. This analysis opens a wide arena for investigation of the novel miRNAs and target pairs, polycistronic miRNAs and the bacterial derived smRNAs predicted in this study.

中文翻译：

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从鹰嘴豆节中鉴定miRNA及其相应的mRNA靶标，并通过在鹰嘴豆根中过度表达来鉴定候选miRNA的功能

豆科植物与根瘤菌建立了共生关系，以满足其对氮的需求。固氮发生在根瘤中，这涉及细菌定植，器官发生和固氮。对一个microRNA和四个RNA末端平行分析（PARE）进行测序，以揭示鹰嘴豆中miRNA介导的共生调控。microRNA文库的测序确定了一组91个miRNA，其中包括84个保守miRNA和7个新颖miRNA。另外，PARE文库分析显示564个基因被85个miRNA靶向。对91个miRNA的前体序列进行的系统进化分析清楚地表明，同一进化枝中两个不同的miRNA聚集在一起，代表着祖先的近亲。此外，使用从不同豆科植物基因组鉴定的miRNA预测了miRNA的生物发生。从结节文库中读取的miRNA也被定位到细菌基因组上，从中可以预测细菌的小RNA。研究了一些miRNA-靶标对的拮抗表达，并且负相关表达谱分析证明了文库和miRNA-靶标对的有效性。基于拮抗表达谱选择四个miRNA，并通过毛状根转化在鹰嘴豆根中异位表达。过表达系显示结节数有明显变化。miR171f（NRK），miR394（HP）和miR1509（AK）的靶标是首次报道的新型靶标。这项分析为研究本研究中预测的新型miRNA和靶对，多顺反子miRNA和细菌衍生的smRNA开辟了广阔的舞台。